Project Summary/Abstract Mucosal surfaces, including those of the ocular surface, are covered by epithelium that is critical to preventing damage and infection. Cell surface-associated mucins are major components on the apical glycocalyx of this epithelium, extending up to 500 nm above the plasma membrane, far above all other membrane-associated proteins and lipids. They exhibit large numbers of O-glycan chains tethered to tandem repeats of amino acids in their protein backbone that create a locally high concentration of carbohydrate ligands. Alteration of cell surface glycosylation has been associated with the disruption of the mucosal barrier in ocular surface epithelial disease (e.g., dry eye), but the mechanisms that lead to this disruption remain mostly uncharacterized. During the last funding period, we identified galectin-3 as the major carbohydrate-binding protein expressed by human ocular surface epithelia. Galectin-3 is a multimeric lectin that can cross-link and oligomerize proteins displaying multiple oligosaccharide chains. This finding led us to hypothesize that multivalent O- glycans bind galectin-3 oligomers to form strong and highly organized lectin-carbohydrate lattices above the undulating membrane ridges in corneal and conjunctival cells' glycocalyx. In this application, we propose to determine whether mucin-type O-glycans in the glycocalyx associate with galectin-3 to form an apical cell surface barrier on the ocular surface, and whether this barrier prevents the influx of foreign substances through the plasma membrane and the access of microorganisms and inflammatory mediators to apical cell surface receptors. Finally, we propose to determine whether in patients with ocular surface epithelial disease there is a molecular breach of the O-glycan:galectin barrier. Understanding the mechanism of epithelial defense that includes components of the cell surface glycocalyx could help with the development of new therapeutic approaches aimed to restore components of the epithelial barrier in patients with ocular surface disease.